Project Summary Heat- and capsaicin-activated ion channel TRPV1 is an important cellular sensor for maintaining normal body temperature and detecting noxious ambient environment, hence an important drug target. TRPV1 also serves as a prototypical model for understanding polymodal activation of diverse TRP channels. Structures of TRPV1 have been solved by cryo-electron microscopy (cyro-EM) at up-to-2.9- resolutions, yet mechanistic understanding of channel activation, in particular by heat, remains preliminary. A major limitation has been the lack of effective tools for probing structural dynamics in functional channels. We have recently identified two structurally related peptide toxins from the venom of Chinese red-headed centipede, termed RhTx1 and RhTx2, that exhibit highly specific and sub-?M affinity binding to the outer pore region of TRPV1. Interestingly, while RhTx1 strongly activates the channel, RhTx2, having just four additional amino acids at its N-terminus, is a potent inhibitor. Our preliminary study further suggested that RhTx1 specifically promotes heat activation. Because these small, compact peptide toxins can be readily synthesized and modified, they offer unique opportunities to probe the molecular mechanism underlying TRPV1 activation, with their rapid ON and OFF kinetics being particularly favorable for biophysical investigation. The proposed study will take advantage of these novel toxins to gain a better understanding of TRPV1 activation mechanism by revealing its structural dynamics using a multidisciplinary approach combines structural, electrophysiological, optical, and computational modeling methods.